Several chemical modifications have been known in all the species of RNAs including messenger RNAs (mRNAs) and other interesting RNAs that are known to regulate translation, such as tRNA, rRNA, and snoRNA. Well-studied examples of common mRNA modification are the 6-adenosine methylation (m6A) and to a lesser extent the 5-cytosine methylation (m5C) and pseudouridylation (Psi). With the advent of new techniques on high-throughput sequencing, it is now much easier to pinpoint such modifications on the mRNA. Such modifications exist in normal cellular tissues as part of the regulatory mechanism of several cellular processes. How this modification reprograms cellular process especially during translation is still a mystery and remains to be investigated. This grant takes advantage of the preliminary studies, showing that there is a change in the start codon selection by chemical modification of mRNA bases. In eukaryotes, the most favored start codon is the AUG followed by CUG, but GUG and UUG remain least favored. However, with the double modification of mRNA by m5C and Psi, the least favored GUG and UUG are more pronounced, while CUG is dampened, leading to the order of preference ? AUG >GUG >UUG >CUG, exactly as observed in prokaryotes. These changes must have paved the way to reprogram translation in response to regulatory cues. Moreover, the threonyl-carbamoylation of the 37th adenosine of the initiator tRNA (t6A), located 3? of its anticodon, has been long proposed to discriminate against GUG initiation in eukaryotes, and more recently postulated to increase the overall initiation accuracy. Here, the preliminary data show that amino acid supply increases non-AUG initiation, while heat increases initiation accuracy and represses non-AUG initiation in a certain yeast strain. Thus, one aim of this proposal is to dissect the ?prokaryotic? start site preference by m5C/Psi mRNA. The preliminary data can be explained if Psi, when introduced to the second position of CUG, GUG and UUG codons, increases initiation from these codons, while m5C, introduced to CUG, dampens the interaction between the anticodon and the CUG codon. These models will be tested by individually modifying reporter mRNA with m5C or Psi and measuring translation from the resulting mRNAs. It is also aimed to verify translation from naturally occurring NPsiG start codons, which were identified by bioinformatics searches. Another aim is to dissect the mechanism of non-AUG translational control by t6A. Molecular dynamics simulations and phenotypic analyses in human and yeast cells will be combined to delineate the molecular basis of translational regulation by chemical RNA modifications.